Research Article

QUALITY STANDARDS OF SAFOOF-E-MUHAZZIL, AN ANTI-OBESITY UNANI FORMULATION

KAMRAN JAVED NAQUVI*1, S. H. ANSARI1, ABUL KALAM NAJMI2

1Department of Pharmacognosy and Phytochemistry, 2Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard, New Delhi 110062, India. Email: kjnaquvi@gmail.com

Received: 20 Mar 2013, Revised and Accepted: 30 Apr 2013

ABSTRACT

Objective: The objective of this study is to establish the quality standards of Safoof-e-Muhazzil (SEMZ), a polyherbal Unani formulation for the treatment of overweight and obesity. Methods: The quality standards of SEMZ were established as per WHO guidelines including heavy metal residues, pesticides residue, aflatoxins, microbial load, GC-MS analysis and HPTLC fingerprinting analysis. Results: The results of standardization parameters showed total ash (10.73±1.23 %), acid-insoluble ash (1.47±0.21%), water-soluble ash (2.91±0.11%), alcohol-soluble extractive (24.95±1.47%), water-soluble extractive (37.46±2.14%), loss on drying (10.03±0.42 %), pH-value for 1% solution (6.01) and 10 % solution (5.3) at 25±2 ºC, fat content (14.13±0.10 %), resin content (9.475±1.2 %) and heavy metals residue, pesticides residue, aflatoxins, microbial load complies with official limits. Conclusion: Data’s evolved in this investigation may be used in laying down Pharmacopoeial standards for the formulation studied, as standardization of herbal medicines as well as polyherbal formulations are absolutely essential and is the need of the hour.

Keywords: Safoof-e-Muhazzil, Standardization, Unani System of Medicine, WHO guidelines.

INTRODUCTION

The World Health Organization (WHO) has estimated that about 80 % of World’s population relies on traditional medicines for primary health care[1]. India is the richest source of medicinal and aromatic plants. Herbal drugs are gaining popularity again day by day in the World since last decades because of its efficacy and lower toxicity as compared to allopathic drugs. Safoof-e-Muhazzil or Sufoof-e-Mohazzil (SEMZ) is a polyherbal formulation used in the Unani System of Medicine for treatment of obesity since ages. The formulation given in National Formulary of Unani Medicine[2] contains ajwain (Nankhwah-Trachyspermum ammi Linn., seeds), ajmoth (Tukhm-e-Karafs-Apium graveolens Linn., seeds), jatamansi (Sumbul-ut-Teeb-Nardostachys jatamansi DC, rhizomes), red rose (Gul-e-Surkh-Rosa damascena Mill., petals), oregano (Marzanjosh- Origanum vulgare Linn., whole plant) and lakh maghsool (Natural resin-animal origin Laccifer lacca Kerr.)[2,3]. Trachyspermum ammi syn. Carum copticum (Apiaceae) commonly known as ajwain, is a glabrous annual plant. Its fruits are aromatic, stems hollow, striated, much branched[4]. Thymol, the major phenolic compound present in ajwain, has been reported to be a germicide, antispasmodic and antifungal agent[5]. Apium graveolens L. (Apiaceae), known as celery, is a common European plant that is now grown, consumed and used as a popular vegetable and spice all over the world. Due to its pleasant odor and nutritional, pharmacological and medicinal values[6,7], this plant product is deemed to be suitable for humans, pets and the environment[8].

Nardostachys jatamansi DC. is a small, perennial, dwarf, hairy, rhizomatous, herbaceous, endangered and most primitive species within family Valerianaceae, distributed in the Himalayas from Pakistan, India (Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Sikkim) to Nepal, Tibet and China between 3300 to 5000 m asl[9]. Rosa damascena Mill belongs to the family Rosaceae, many species and variety of which are cultivated throughout the world as an ornamental plant[10,11]. Origanum vulgare L. belonging to mint family Lamiaceae, is an aromatic plant with a wide distribution throughout the Mediterranean area and Asia[12]. Major components are carvacrol and thymol that constitute about 78 to 82% of the total oil[13]. Lac (Lakh Maghsool) is the only natural resin of animal origin, produced by a red coloured tiny Lac insect, Laccifer lacca (Kerr) found throughout India. Fourteen species of insect Laccifer are reported in India on a number of plants both wild and cultivated in various regions[14]. The present study deals with the evaluation of quality parameters of SEMZ.

MATERIAL AND METHODS

Plant Material and Authentication

All the drugs were purchased from Samsi Dawakhana, Ballimaran, Delhi and authenticated by Dr. H.B. Singh, Scientist F and Head, Raw Materials Herbarium and Museum, National Institute of Science Communication and Information Resources (NISCAIR), New Delhi. Voucher specimens of drugs were deposited in the Raw Materials Herbarium and Museum, National Institute of Science Communication and Information Resources (NISCAIR), New Delhi, with reference number Ref. NISCAIR/RHMD/consult/-2010-11/1705/05.

Preparation of Formulation

All the drugs were dried in shade and powdered and passed through sieve no. 100. The formulation was prepared as the method described in National Formulary of Unani Medicines[2]:

1. Ajwain (Tachyspermum ammi L.)Seeds10 g.

2. Ajmoth (Apium graveolens) Seeds 10 g.

3. Jatamansi (Nardostachys jatamansi) Roots10 g.

4. Luk Maghsool (Purified Laccifer lacca)Resin10 g.

5. Gul-e-Surkh (Rosa damascena) Petals 25 g

6. Marzanjosh (Origanum vulgare) Whole plant 25 g

Morphological studies

Proper examination of the each ingredients of the SEMZ was carried out under diffused sunlight and artificial source similar to day light (Fig 1)[15].

Powder microscopy

The microscopic examination of powdered SEMZ was performed. Slides of powdered SEMZ were prepared and stained with chloral hydrated, phloroglucinol and HCl. Microphotography on different magnifications was carried out with Motic microscopic unit (Hong Kong). Polarized light was used for the study of crystals, starch granules and lignified cell[15].

Physico-chemical standardization

The various physico-chemical values of SEMZ such as ash values (Table 1), extractive values[16] (Table 2), loss on drying were determined according to the Pharmacopoeial method[17].

International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 5, Suppl 2, 2013

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Powdered drug reaction with different reagents

The powdered SEMZ was treated separately with different reagents and acids like, picric acid, hydrochloric acid, nitric acid, iodine, ferric chloride, and sodium hydroxide the colour shown by that treatment is noted as such and under the microscope[18] (Table 3).

Fluorescence Analysis

Many herbs fluorescence when cut surface or powder is exposed to UV light and this can help in their identification method. The fluorescence character of the SEMZ powder (40 mesh) was studied both in daylight and UV light (254 and 366 nm) and after treatment with different reagents like sodium hydroxide, picric acid, acetic acid, hydrochloric acid, nitric acid, iodine, ferric chloride etc (Table 4)[19,20].

Phytochemical screening

The phytochemical evaluation of SEMZ was carried out as per the method described[21]. Powdered SEMZ (5 g) were extracted in a Soxhlet apparatus with petroleum ether, chloroform, acetone, alcohol and water. The extracts were evaporated to dryness under vacuum. These extract were used for the analysis of different phyto-constituents viz. alkaloids, carbohydrates, phenolics, flavonoids, proteins, amino acids, saponins, and resins etc (Table 5)[16].

Loss on drying

The powdered SEMZ (10 gm) without preliminary drying was placed on a tarred evaporating dish and dried at 105 ºC for 6 hours and weighed. The drying was continued until two successive reading matches each other or constant weight was reached when two consecutive weighing after drying for 30 minutes in a desiccator, showed not more than 0.01 gm difference (Table 1)[22].

Determination of pH

The pH of 1 % and 10 % aqueous solution of SEMZ were checked (Table 1) by standardized glass electrode (Hanna pH meter, USA)[23].

Determination of fat content

A weighed quantity of SEMZ sample (3 g) is extracted with anhydrous ether in a continuous extraction apparatus for six hours the extract is filtered into a clean dry weighed flask. The extraction flask is rinsed with small quantity of ether, filtered and added to the weighed flask. The solvent is evaporated and dried to constant weight at 105 ºC (Table 1)[24].

Determination of resin content

The accurately weighed SEMZ sample (5 g) was rapidly refluxed with acetone (3 X 200 ml) for 6 h to exhaust the drug for the resin content. The excess solvent was removed by distillation on a water

bath. The residue so obtained was suspended in water and transferred to a separating funnel, repeatedly extracted the suspension with solvent ether (2 X 200 ml) to extract all the resin contents. The ether extracts were cooled out dried over anhydrous sodium sulphate and excess ether removed over a water bath. It was transferred to a weighed beaker and the final weight is noted (Table 1)[24].

Determination of microbial load (Table 6, fig 3), heavy metal residues (Table 7) and pesticides residue contents (Table 8) and aflatoxins (Table 9) were also done as per WHO guidelines[15].

GC-MS analysis of SEMZ

The analysis of the volatile constituents were run on a Shimadzu QP-2010 GC-MS system equipped with AB-Innowax 7031428 WCOT column (60 m x 0.25 mm x 0.25 μm) directly coupled to the MS. The carrier gas was helium with a flow rate of 1.21 ml/min oven temperature was programmed as 50 ºC for 1 min and subsequently held isothermal for 2 min injector port: 250 ºC, detector: 280 ºC, split ratio 1: 50, volume injected: 1 μl of the oil. The recording was performed at 70 eV, scan time 1.5 s; mass range 40-750 amu. Software adopted to handle mass spectra and chromatograph was a Chem station. Further identification was made by comparison of fragmentation pattern of mass spectra obtained by GC-MS analysis with those stored in the spectrometer database of NBS 54 K.L, WILEY 8 libraries and published literature[25-30].

HPTLC fingerprinting analysis of SEMZ

The weighed quantity (20 g) of SEMZ was extracted in a Soxhlet apparatus for 6 h using twice the amount of solvent (hexane) at a controlled temperature. The dried extract was dissolved in the respective solvent (2 mg/ml). HPTLC was performed on 5 cm × 10 cm aluminum backed plates coated with silica gel 60F254 (Merck, Mumbai, India). Standard solution of thymol and sample solution were applied to the plates as bands 5.0 mm wide of the same chromatographic plate by use of a Camag (Muttenz, Switzerland) Linomat V sample applicator equipped with a 100 μl Hamilton (USA) syringe. Ascending development to a distance of 80 mm was performed at room temperature (28 ± 2°C), with toluene: ethyl acetate, 9 : 1 (v/v), as mobile phase, in a Camag glass twin-trough chamber previously saturated with mobile phase vapour for 20 min. After development, the plates were dried and sprayed with anasaldehyde-sulphuric acid reagent and then scanned at 513 nm with a Camag TLC Scanner with WINCAT software, using the deuterium lamp (Fig.3) [31].

RESULTS AND DISCUSSION

Morphological characters

Proper examination of the untreated samples of ingredients of SEMZ was carried out under diffused sunlight and artificial source similar to day light (Fig. 1).

Trachyspermum ammi Apium graveolens Nardostachys jatamansi

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Rosa damascena Origanum vulgare Lakh resin

Fig. 1: Ingredients of Safoof-e-Muhazzil

Organoleptic characteristics of SEMZ powder

Colour: Dark brown

Odour: Characteristics, aromatic

Taste: Acrid

Powder microscopy

Fig. 2: Powder characteristics of Safoof-e-Muhazzil

a. Trichomes, parenchymatous cells, oil globules, calcium oxalate crystals; b. Parenchymatous cells of R. damanscena petals; c. Tangentially cut medullary rays of N. jatamansi; d. Calcium oxalate crystals; e. sclerenchymatous cells with oil glands; f. Endocarp of seeds; g. Fibre; h. Broken trichome; i. Cortex with vascular bundle; j. Xylem vessel; k. Stone cells.

a

b

c

d

e

f

g

h

i

j

k

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Table 1: Physico-chemical parameters

Parameters Values (% Mean ± SEM) Total ash value 10.73 ± 1.23 Acid insoluble ash 1.47 ± 0.21 Water soluble ash 2.91 ± 0.11 Loss on drying 10.03 ± 0.42 Resin content 9.475 ± 1.2 Fat content 14.13 ± 0.10 pH values

6.1 (1 % solution) 5.3 (10 % solution)

Extractive values

The extractive values of SEMZ were done as per procedure described above. All the procedure was repeated in triplicate (Table 1). The mean values ± SEM are given below:

Table 2: Extractive values of SEMZ

Extraction Extractive value, Mean (%) ± SEM Petroleum ether Chloroform Acetone Alcoholic Hydro alcoholic Aqueous Hot 8.83 ±0.72 4.86 ± 0.38 14.68±1.26 18.68± 2.73 21.36 ±1.37 23.18±1.52 Cold 7.07± 0.47 5.81 ± 0.18 7.81 ±0.38 17.10± 1.38 24.12±1.72 25.97± 1.12 Successive 5.67± 0.82 14.05± 0.94 16.92±0.75 24.95±1.47 44.65± 2.38 37.46±2.03

Table 3: Powdered drug reaction with different reagents

The powder of SEMZ was treated with different reagents which showed following colour:

Chemical treatment Observation Chemical treatment Observation Iodine Dark brown Sodium hydroxide (5%) Light brown 50% HNO3 Orange Glacial acetic acid Brown 1N H2SO4 Crimson red Ferric chloride (5%) Black Lead acetate Light green 1N HCL Brown Picric acid Yellow KOH (1%) Orange

Table 4: Fluorescence analysis of SEMZ

The powder of SEMZ (mesh size 40) was examined under daylight and UV light. The observation was recorded as under:

Treatment with Day light UV light (254 nm) UV (366 nm) Powder as such Light brown Light brown Dark brown Distilled water Light brown Brown Black 5% NaOH Brownish red Black Black H2SO4 Dark brown Dark brown Black Conc. HCL Dark brown Black Black Acetone Brown Reddish brown Dark brown Chloroform Greenish yellow Dark brown Black Conc. HNO3 Yellowish red Dark brown Black Methanol Yellowish green Red Light green FeCl3 Grey Brown Black

Table 5: Results of phytochemical screening of SEMZ

Extract constituents Petroleum ether Chloroform Acetone Alcoholic Hydro-alcoholic Aqueous Alkaloids - + - + + + Glycosides - + + + ++ + Tannin + ++ ++ ++ +++ +++ Phenolics + ++ + +++ ++ ++ Flavonoids - + ++ +++ ++ ++ Proteins - - - + + + Saponins - - - + + + Resins + + ++ + - - Sugars + + + ++ ++ +++ Lipids/fats ++ + + + - -

(+++) Strongly Positive, (+) Positive test, (-) Negative test

Table 6: Microbial load

Colony forming units on nutrient agar medium.

Test Result Total plate count cfu/ g 23181 Yeast/mould 886

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Table 7: Heavy metal content of SEMZ

Heavy metal Heavy metal in the formulation

Pharmacopoeial percentage limit of heavy metals in crude drugs

Lead Not Detected Not more than 10.0 ppm Cadmium Not Detected Not more than 0.3 ppm Mercury Not Detected Not more than 1.0 ppm Arsenic Not Detected Not more than 10.0 ppm

Table 8: Pesticides residue of SEMZ

Pesticides Test Method Result MDL γ-BHC(Lindane) AOAC 970.52/EPA 525.2 Not Detected 0.01 mg/kg δ-BHC AOAC 970.52/EPA 525.2 Not Detected 0.01 mg/kg Heptachlor AOAC 970.52/EPA 525.2 Not Detected 0.01 mg/kg Chlorpyrifos AOAC 970.52/EPA 525.2 0.3 mg/kg 0.01 mg/kg α-Chlordane AOAC 970.52/EPA 525.2 Not Detected 0.01 mg/kg α-Endoulfan AOAC 970.52/EPA 525.2 Not Detected 0.01 mg/kg β-Chlordance AOAC 970.52/EPA 525.2 Not Detected 0.01 mg/kg Endrin AOAC 970.52/EPA 525.2 Not Detected 0.01 mg/kg Total DDT AOAC 970.52/EPA 525.2 Not Detected 0.01 mg/kg Aldrin AOAC 970.52/EPA 525.2 Not Detected 0.01 mg/kg Methoxychlor AOAC 970.52/EPA 525.2 Not Detected 0.01 mg/kg Dieldrin AOAC 970.52/EPA 525.2 Not Detected 0.01 mg/kg

Aflatoxins

Aflatoxin B1, Aflatoxin B2, Aflatoxin G1 and Aflatoxin G2 was found below the detection limit while minimum detection limit was 1.0 µg/kg.

GC-MS analysis

Fifty eight compounds representing 99.35 % of the oil were characterized. Consisting of thirteen monoterpenes (11.62 %), forty four sesquiterpenes (85.05 %), two diterpene alcohols (0.56 %),

trans-phytol and 13(16),14-labdiene-8-ol, four aliphatic hydrocarbons n-eicos-15-enol, 9,12-octadecadienoic acid methyl ester, n-heptacosane and methyl-6-hydroxyoctadecanoate and two aromatic components 2-benzilidine and amyrolin. Eudes-4(14),11-diene (28.61 %), viridiflorol laurate (16.40 %), bisabolene (9.73 %), globulol (9.13 %), thymol (6.14 %), t-cadinol (4.15 %), trans-cadine-1,4-diene (2.08 %), 2E, 6E-farnesol (1.41), L-limonene (1.39 %), -cadinene (1.37 %) and β-gurjunene (1.28 %) were the predominant compounds[3].

HPTLC fingerprinting analysis

Table 9: TLC Details of Test Solution of SEMZ after derivatization with anasaldehyde-sulphuric acid reagent.

SEMZ oil SEMZ Hexane extract Rf Value Colour of the band Rf Value Colour of the band 0.10 Pink 0.06 Violet 0.19 Faint purple 0.13 Light pink 0.25 Faint purple 0.22 Light purple 0.27 Light blue 0.33 Light blue 0.29 Orange 0.40 Pink 0.35 violet 0.46 Light brown 0.40 Light brown 0.58 Orange (Thymol) 0.44 Light blue 0.65 Purple 0.58 Orange (Thymol) 0.75 Light brown 0.62 Pink 0.69 Brown 0.74 Light brown

1 2 3 4

Fig. 3: TLC profile of test solution of SEMZ after derivatization with anasaldehyde-sulphuric acid reagent.

1: Thymol standard; 2-3: SEMZ oil; 4: Hexane extract.

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CONCLUSION

The present study is related to pharmacognostical, physicochemical and preliminary phytochemical screening of Safoof-e-Muhazzil. Phytochemical study is also useful to isolate the pharmacologically active principles present in the drugs. This work may be used as standard monograph for identification and evaluation of the other such formulations[32,33].

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